1. Field of the Invention
The present invention relates to novel human growth factors. More specifically, isolated nucleic acid molecules are provided encoding huXAG-1, huXAG-2, and huXAG-3. HuXAG-1, huXAG-2, and huXAG-3 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of huXAG-1, huXAG-2, and huXAG-3 activity, as well as diagnostic and therapeutic uses of huXAG-1, huXAG-2 and huXAG-3.
2. Related Art
Growth Factors
Control of cell division is a basic aspect of multicellular existence that depends upon a programmed series of events. One factor in cellular proliferation and its control that has been identified is the presence of various polypeptide growth factors. Growth factors are essential components of growth media for in vitro cell culture and are involved in cell survival in vivo. Some of the growth factors that have been identified to date include PDGF (platelet-derived growth factor) implicated in the repair of the vascular system in vivo; EGF (epidermal growth factor) which acts as a mitogen for cells of ectodermal and mesodermal origin; TGF-xcex1 (transforming growth factor) which acts as a mitogen similarly to EGF but can make normal cells grow in agar, TGF-xcex2 (transforming growth factor) which is a mitogen for some cells and a growth inhibitor for others; and NGF (nerve growth factor) involved in the development and maintenance of sympathetic and embryonic neurons. Watson et al., Molecular Biology of the Gene, p. 975 (Benjamin/Cummings 1987).
It is clear that particular cell types require particular growth factors. Peptide growth factors are produced and secreted from a variety of tissues. The target cells are typically located close to the site of release of the growth factor (paracrine response). In addition to their growth promoting and differentiation-inducing activities, growth factors elicit a wide variety of effects in their target cells and are involved in processes such as inflammation, immune reactions and wound repair. See, Pimentel, E. Handbook of Growth Factors, Volume 1: General Basics (CRC Press 1994).
Monitoring of growth factor gene expression in cells of various human tissue, would be useful in detecting and studying abnormal hypertrophy both in vitro and in vivo. In addition, monitoring for higher than normal expression of tissue-specific growth factors would be useful in early diagnosis of certain cancers.
Additionally, polypeptide growth factors are very important cell culture reagents for stimulating cellular growth and aiding survival of the cells in vitro.
The search continues to exist for polypeptides that stimulate and/or inhibit growth of particular cells for both in vitro and in vivo uses. In addition, the search continues for novel tissue specific markers that can be employed qualitatively to help identify a particular cell or tissue type and employed qualitatively to assess whether cells, tissues or organs are abnormal in their expression of a particular polypeptide.
At the front of the larvae in most frog species lies a small and sticky patch of cells, called the cement gland. The cement gland lies anterior to the brain and any other neural tissue and develops from the embryonic ectoderm (Drysdale et al., Dev. Growth Differ. 34:51-59 (1992)). Formation of the cement gland is influenced by multiple signals (Sive et al., Dev. Dyn. 205:265-280 (1996)). One such signal is the XAG protein, which is expressed at very high levels in the cement gland and at lower levels in the more posterior hatching gland. It encodes a secreted protein later expressed in the pharynx (Jamrich et al., Development 105:779-786 (1989)) and in the lung primordium.
Colorectal Carcinoma
Colorectal carcinoma is a cancer which affects many people per year. The prognosis is poor in about 50% of the cases because the tumor is often not detected until the disease has spread and has reached a terminal stage. Early diagnosis is important to increase chances of arresting the carcinoma before it metastasizes, thereby leading to an improved prognosis.
One method of early tumor diagnosis is detection of the presence of a marker or antigen specific for a particular tumor. These normally proteinaceous markers are synthesized by the tumor, and may be found in the serum and/or on the surface of the tumor. Only a limited number of tumor markers for colorectal carcinoma have thus far been found to have clinical use. These include carcinoembryonic antigen (CEA), and the sialyated Lewis a antigen (CA 19.9). Unfortunately, approximately 40% of patients whose condition has been accurately diagnosed as colorectal carcinoma do not have elevated plasma levels of either of these antigens when initially examined. There is a need for commercially available serodiagnostic markers which can be used to detect the tumor and to monitor therapy for this group.
Another method of diagnosis involves detecting the presence of a gene associated with a particular disease or condition. A need exists for genetic markers which can be detected by genetic screening tests for the early detection of colon cancer in high risk patients.
Accordingly, it is an object of this invention to provide a new marker for the detection of colorectal carcinoma.
It is another object of the invention to provide a new marker suitable for diagnosing and monitoring colorectal cancer. Such novel marker can be further used to make novel antibodies or other antagonists that bind the colon cancer specific polypeptides to diagnose cancer and to bind and inhibit the biological function of the polypeptide.
A further object of the present invention is to provide a method and kit for the detection and monitoring of colorectal carcinoma in patients using assay methods specific for markers associated with colorectal carcinoma cells.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the huXAG-1 (also referred to as colon cancer-specific gene (CCSG)) polypeptide having the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 97641 on Jun. 27, 1996. The nucleotide sequence was determined by sequencing the deposited huXAG-1 clone and is shown in SEQ ID NO:1. The sequence contains an open reading frame encoding a polypeptide of about 175 amino acid residues, with a leader sequence of about 20 amino acid residues, and a predicted molecular weight of about 20 kDa. The amino acid sequence of mature huXAG-1 is shown in SEQ ID NO:2 (amino acid residues from about 1 to about 155 in SEQ ID NO:2).
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the huXAG-1 polypeptide having the complete amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequence encoding the huXAG-1 polypeptide having the complete amino acid sequence in SEQ ID NO:2 but minus the N-terminal methionine residue; (c) a nucleotide sequence encoding the mature huXAG-1 having the amino acid sequence at positions from about 1 to about 155 in SEQ ID NO:2; (d) a nucleotide sequence encoding the huXAG-1 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97641; (e) a nucleotide sequence encoding the mature huXAG-1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97641; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e) above.
The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the huXAG-2 polypeptide having the amino acid sequence shown in SEQ ID NO:4 or the amino acid sequence encoded by the cDNA deposited as ATCC Deposit Number 209134 on Jun. 27, 1997. The nucleotide sequence determined by sequencing the deposited huXAG-2 clone, which is shown in SEQ ID NO:3, contains an open reading frame encoding a polypeptide of about 172 amino acid residues, with a leader sequence of about 23 amino acid residues. The amino acid sequence of the mature huXAG-2 is shown in SEQ ID NO:4 (amino acid residues from about 1 to about 149 in SEQ ID NO:4).
Thus, another aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the huXAG-2 polypeptide having the complete amino acid sequence in SEQ ID NO:4; (b) a nucleotide sequence encoding the huXAG-2 polypeptide having the complete amino acid sequence in SEQ ID NO:4 but minus the N-terminal methionine residue; (c) a nucleotide sequence encoding the mature huXAG-2 having the amino acid sequence at positions from about 1 to about 149 in SEQ ID NO:4; (d) a nucleotide sequence encoding the huXAG-2 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209134; (e) a nucleotide sequence encoding the mature huXAG-2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209134; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e) above.
The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the huXAG-3 polypeptide having the amino acid sequence shown in SEQ ID NO:6 or the amino acid sequence encoded by the cDNA deposited as ATCC Deposit Number 209137 on Jul. 3, 1997. The nucleotide sequence determined by sequencing the deposited huXAG-3 clone, which is shown in SEQ ID NO:5, contains an open reading frame encoding a polypeptide of about 166 amino acid residues, with a leader sequence of about 23 amino acid residues. The amino acid sequence of the mature huXAG-3 receptor is shown in SEQ ID NO:6 (amino acid residues from about 1 to about 143 in SEQ ID NO:6).
Thus, another aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the huXAG-3 polypeptide having the complete amino acid sequence in SEQ ID NO:6; (b) a nucleotide sequence encoding the huXAG-3 polypeptide having the complete amino acid sequence in SEQ ID NO:6 but minus the N-terminal methionine residue; (c) a nucleotide sequence encoding the mature huXAG-3 having the amino acid sequence at positions from about 1 to about 143 in SEQ ID NO:6; (d) a nucleotide sequence encoding the huXAG-3 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209137 ; (e) a nucleotide sequence encoding the mature huXAG-3 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209137; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences of huXAG-1, huXAG-2 or huXAG-3 in (a), (b), (c), (d), (e), or (f), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e), or (f), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a huXAG-1, huXAG-2, or huXAG-3 polypeptide having an amino acid sequence in (a), (b), (c), (d), or (e), above.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of huXAG-1, huXAG-2, or huXAG-3 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated huXAG-1 polypeptide having an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the huXAG-l polypeptide having the complete 175 amino acid sequence, including the leader sequence shown in SEQ ID NO:2; (b) the amino acid sequence of the huXAG-1 polypeptide having the complete 175 amino acid sequence, including the leader sequence shown in SEQ ID NO:2 but minus the N-terminal methionine residue; (c) the amino acid sequence of the mature huXAG-1 polypeptide (without the leader) having the amino acid sequence at positions 1-155 in SEQ ID NO:2; (d) the amino acid sequence of the huXAG-1 polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No. 97641; and (e) the amino acid sequence of the mature huXAG-1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97641.
The invention further provides an isolated huXAG-2 polypeptide having an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the huXAG-2 polypeptide having the complete 172 amino acid sequence, including the leader sequence shown in SEQ ID NO:4; (b) the amino acid sequence of the huYAG-2 polypeptide having the complete 172 amino acid sequence, including the leader sequence shown in SEQ ID NO:4 but minus the N-terminal methionine residue; (c) the amino acid sequence of the mature huXAG-2 polypeptide (without the leader) having the amino acid sequence at positions 1-149 in SEQ ID NO:4; (d) the amino acid sequence of the huXAG-2 polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No. 209134; and (e) the amino acid sequence of the mature huXAG-2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209134.
The invention also provides an isolated huXAG-3 polypeptide having an amino acid sequence selected from the group consisting of (a) the amino acid sequence of the huXAG-3 polypeptide having the complete 166 amino acid sequence, including the leader sequence shown in SEQ ID NO:6; (b) the amino acid sequence of the huXAG-3 polypeptide having the complete 166 amino acid sequence, including the leader sequence shown in SEQ ID NO:6 but minus the N-terminal methionine residue; (c) the amino acid sequence of the mature huXAG-3 polypeptide (without the leader) having the amino acid sequence at positions 1-143 in SEQ ID NO:6; (d) the amino acid sequence of the huXAG-3 polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No. 209137; and (e) the amino acid sequence of the mature huXAG-3 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209137.
The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical to the huXAG-1, huXAG-2 or huXAG-3 polypeptides described above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a huXAG-1, huXAG-2 or huXAG-3 polypeptide having an amino acid sequence described in (a), (b), (c), (d), or (e), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a huXAG-1, huXAG-2 or huXAG-3 polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids. It is understood that epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention. In another embodiment, the invention provides an isolated antibody that binds specifically to a huXAG-1, huXAG-2 or huXAG-3 polypeptide having an amino acid sequence described in (a), (b), (c), (d), or (e) above.
The invention further provides methods for isolating antibodies that bind specifically to a huXAG-1, huXAG-2 or huXAG-3 polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as describe below.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by huXAG-1, huXAG-2 or huXAG-3 protein which involves contacting cells which express huXAG-1, huXAG-2 or huXAG-3 protein with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
A method for detecting and monitoring human colorectal carcinoma is based upon the discovery of the gene encoding huXAG-1. This protein was found to be expressed in cells derived from a human colon carcinoma, but not in other human tissues. In a first embodiment the method measures the level of huXAG-1 mRNA expression in a biological sample, preferably a tissue biopsy, and compares the measurement to a standard value. Levels of mRNA expression may be assayed by a variety of well-known methods, including Northern blot analysis, S1 nuclease mapping and polymerase chain reaction (PCR).
In a second embodiment, the method for detecting and monitoring human colorectal carcinoma includes contacting a biological sample with an antibody or portion thereof which reacts with huXAG-1, an analog, or portion thereof, and observing if the antibody reacts with the sample. The biological sample may be whole blood, serum, ascites fluid, a tissue biopsy, a tumor, a tissue culture, or a histological preparation thereof The antibody may be raised to huXAG-1 polypeptide, or to analogs or antigenic portions thereof. More specifically, this antibody may be a polyclonal or monoclonal antibody, or analog or portion thereof, which preferably does not cross-react with CEA, NCA, CA 19.9, alpha-1-acid glycoprotein, or with the blood group substances A, B, and H. An immunoassay may be utilized to observe the extent of reaction between the receptor and the sample.
A detection method is also contemplated which enables the diagnosis and identification of a tumor cell in a biological sample from a patient. In this method the sample is subjected to at least one of a plurality of tests, each of which is specific for a particular tumor marker. The test may be any type of assay, preferably an immunoassay which employs an antibody specific for a tumor marker. The tests may be carried out sequentially until one of them indicates the presence of a particular marker.
Further, a kit for screening a patient for colorectal carcinoma is contemplated which contains a plurality of different antibodies specific for tumor markers. These antibodies may include those reacting with one of the following markers: huXAG-1; CEA; NCA; CA 19.9; and alpha-1-acid glycoprotein.
A method for diagnosis of diseases related to altered levels of huXAG-1, huXAG-2 or huXAG-3 gene expression is also provided,
Also provided are methods for identifying receptors of huXAG-1, huXAG-2 or huXAG-3.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of huXAG-1, huXAG-2 or huXAG-3 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated huXAG-1, huXAG-2, or huXAG-3 polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of huXAG-1, huXAG-2, or huXAG-3 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an huXAG-1, huXAG-2, or huXAG-3 antagonist. Preferred antagonists for use in the present invention are huXAG-1-, huXAG-2-, or huXAG-3-specific antibodies.